The Study of IL-10 and IL-17A Genes Expression in Patients with Different Stages of Asthma: a Case-Control Study.

Background
Asthma is considered as a complex disorder in which genetics and environment play crucial role in its susceptibility. In addition to the huge financial costs that significantly reduce the quality of life of the patients and their families, it causes high prevalence of lung diseases. Finding contributing new genetic factors involved in early diagnosis or progression of asthma can provide novel approaches for treatment or managing of asthma. In the present study, the potential role of two key cytokines of IL-10 and IL-17A was investigated in asthma pathogenesis.


Materials and Methods
Using real-time PCR technique, we analyzed the expression levels of target genes in two groups of mild and severe asthma patient in comparison with healthy individuals.


Results
In comparison with control population, obtained data showed 4 and 7-fold down-regulation of IL-17A in the group of mild and severe asthma, respectively. Down-regulation of IL-17A showed a significant correlation with progression of asthma severity. While IL-10 showed up to 10-fold down-regulation in the group of severe asthma, its expression level was not correlated with severity of asthma.


Conclusion
Obtained data revealed that deregulation IL-10 and IL-17A have potential to play crucial role in pathogenesis and prognosis of asthma. Observed down-regulation of these cytokines in blood cells suggests their usefulness as a marker in diagnosis of asthmatic types in patients.


INTRODUCTION
Asthma is, a common complex disease, characterized by chronic inflammation of airway, variable airflow limitation, repetitive cough, wheezing, shortness of breath and chest tightness (1). Asthma is associated with considerable morbidity, avoidable mortality and substantial costs to society (2). According to the estimation of World Health Organization (WHO) more than 300 million people currently suffer from asthma worldwide and this number is expected to grow to 400 million by 2025 (3). Although environmental factors play key role in the pathogenesis of asthma, there are multiple reported genes which confer susceptibility to this disease (5,6). Owing to complexity, it has been difficult to identify the genetic basis of such complex genetic disorders and yet, the genetic contribution of asthma disease is remained to be investigated (7). Accumulating evidence suggest that immune system cells such as TH17 cells and their related cytokines are involved in the pathophysiology of asthma (8,9). These data emphasize the prominent role of cytokines and their receptors in promotion of allergy and asthma (10)(11)(12).
IL-10 is a cytokine derived from CD4 + T-helper type 2 (TH2) cells (13,14) identified as a suppressor of cytokines from T-helper type 1 (TH1) cells (15,16). IL-10 contributes in the pathophysiologic mechanism of inflammatory disease since it has been shown to regulate both cellular and humoral immunity (17).
Distinct responses of IL-17-producing cells in inflammatory conditions (18) together with its increase in lung lesions (19,20), highlighted the potential involvement of IL-17 in asthma (21) and potentiality it could be a modifier gene specific to asthma. In addition, IL-17 family   members including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F (22,23) are known to be important regulator of neutrophilic inflammation (24) and aberration in their production may drive severe forms of the disease (21,25).
Among IL-17 family members, the expression patterns and function of IL-17A is not well understood.
In this study, we evaluated the expression alteration of Interleukin-10 (IL-10) and Interleukin-17A (IL-17A) in some categories of asthma patients with different level of severity versus healthy controls.   To examine genes expression of interleukins 17A (IL- expression level in normal control population (Figure 2A).

RNA preparation of Peripheral Blood Mononuclear Cells
In addition, to assess correlation in normal population, this parameter was calculated for patients and results showed that unlike absence of correlation in patients with mild asthma (r=0.451; p-value=0.106) ( Figure 2B), there was a significant correlation (r=0.790; p-value=0.001) between IL-10 and IL-17A transcription level in severe asthma ( Figure   2C).

Lack of association between IL-10 and IL-17A expression level and age
To check if there was an association between expression level of IL-10 and IL-17A, correlation co-efficient was calculated separately in normal, mild and severe populations. Data revealed that there was not any significant association between the expression level of these genes and age (Figures 3 A, B, C, D, E, and F).

DISCUSSION
A growing amount of evidence suggests that different types of asthma such as the severe type could be associated with interleukins' production and accumulation (12). Of those, IL-17 may be one of the major cytokines involved in exacerbation of bronchial asthma (26). IL-17A is one of the IL-17 family members and it is considered as a proinflammatory cytokine, playing important role in the induction and propagation of different immunological symptoms such as alveolar inflammation (27). IL-10, as an inhibitor of cytokines from T-helper type 1 cells, is a cytokine derived from CD4+ T-helper type 2 cells (28,29).
The role of IL-10 in asthma pathogenesis has not been well documented. Some evidence suggests that the production of IL-10 is enhanced in the bronchial mucosa of asthmatic subjects in comparison to non-asthmatic control subjects (30).
Although many genes (including cytokine genes) have been identified or suspected to be involved in the IL-10 is a pluripotent cytokine with widely distribution that plays a dual role in inflammation. Decreasing IL-10 production is accompanied by the increase of proinflammatory cytokines' production which consequently leads to chronic inflammation, airways remodeling, airflow obstruction and lung tissue damage (29). Polymorphisms in the gene of IL-10 has been defined to play a substantial role in the inflammatory response during the onset of asthma (42,43).
Our findings showed a significant decreased level of IL-those with mild asthma and controls ( Figures 1B and C). However, difference in expression level of IL-10 between mild asthmatic patients and healthy individuals was statistically significant. This data suggests that because the change of IL-10 expression is limited to late stages of asthma, it could be useful for prognostic marker of asthma.
Since attenuated production of intracellular IL-17A and IL-10 in mononuclear cells from patients with severe asthma could not be explained by progression of asthma stages, we decided to investigate if the expression levels of are not probably independent. This suggests that there may be interplay between the two cytokines in the pathogenesis of asthma and their correlated expression ablation contribute in asthma progression. However, lack of significant correlation between IL-10 and IL-17A in mild asthma patients suggests different function of these cytokines during initiation of asthma which environment is considered as a causal role (44,45). Consistently, we found that the IL-10 expression level is not correlated to the severity of asthma and other factors may be contributing.
Further studies of larger patient cohorts and blood samples are needed to confirm and explain these findings and study of correlation between expression and the protein of target genes would be interesting.